CN113813300A - Preparation method and application of glycyrrhiza glabra extract - Google Patents

Preparation method and application of glycyrrhiza glabra extract Download PDF

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CN113813300A
CN113813300A CN202111287796.XA CN202111287796A CN113813300A CN 113813300 A CN113813300 A CN 113813300A CN 202111287796 A CN202111287796 A CN 202111287796A CN 113813300 A CN113813300 A CN 113813300A
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glycyrrhiza glabra
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ethanol
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ethyl acetate
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CN113813300B (en
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贺飞
信学雷
吴涛
程珍
刘柳
李淑萍
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Xinjiang Technical Institute of Physics and Chemistry of CAS
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Abstract

The invention relates to a preparation method and application of a glycyrrhiza glabra extract, wherein the extract is prepared by reflux extracting organic solvent ethanol, separating and purifying macroporous resin, settling petroleum ether and ethyl acetate to obtain effective components for reducing blood sugar, wherein the content of glabridin is 40-70%, and preliminary activity screening tests prove that: the effective part obtained by the method has the inhibition effect on protein tyrosine phosphatase 1B (PTP-1B) and alpha-glucosidase, and can be used as a raw material or an auxiliary agent for preparing a medicament or a health-care product for preventing and treating diabetes. The method has the advantages of simple process, low production cost, stable quality of effective components and easy control.

Description

Preparation method and application of glycyrrhiza glabra extract
Technical Field
The invention relates to a preparation method of a glycyrrhiza glabra extract and application of the extract in medicines, health-care products or other functional foods for reducing blood sugar.
Background
The liquorice is an important bulk traditional Chinese medicine, has the effects of tonifying spleen and qi, clearing heat and removing toxicity, eliminating phlegm and stopping cough, relieving spasm and pain and harmonizing the medicines, and is widely applied in traditional Chinese medicine formulas. Statistics show that the demand of liquorice is at the top of a large number of medicinal materials and is increased year by year, but the liquorice industry still takes crude processing industries such as medicinal material decoction pieces and extracts as main materials at present, the deep processing technology of liquorice products falls behind, the industrial scale is small, and the extension of the liquorice industry chain and the development of the liquorice industry are severely limited.
Glycyrrhiza glabra L. is a plant of the genus Glycyrrhiza and has multiple functional components such as glycyrrhizic acid and glabridin. Glabridin has excellent effects of resisting inflammation, oxidation and melanin formation, is known as whitening gold in the world cosmetics industry, is a main functional component of international high-grade whitening cosmetics, and is a whitening product of brands such as ashilan, lancome, rain and Helianna. The glabridin is sold in the market by a conventional method, but the cost of the glabridin for preparing skin care products is very high due to the rare content and the great extraction difficulty.
The extraction and purification method of glabridin comprises recrystallization, silica gel column chromatography, resin method, etc. The patent CN107510710A adopts macroporous resin and one or two of polyamide and MCI to prepare licorice total flavone, mainly prepares licorice chalcone compound, wherein chalcone A accounts for 10-20% of the extract content, because polyamide and MCI have smaller grain diameter, the column chromatography is easy to block, and the use is not much in practical production. In patent CN112778323A, high-purity glabridin is obtained by ethanol extraction, macroporous resin purification and reversed phase silica gel column chromatography, but the process period is long and the consumption of organic solvents is high. Patent CN105777771A adopts silica gel column chromatography and recrystallization to obtain glabridin, and has the problems of high consumption of silica gel and organic solvent, environmental pollution and the like.
With the progress of society and the improvement of living standard of people, the number of diabetics, especially type II diabetes (non-insulin dependent diabetes) is increased by times. Protein Tyrosine phosphatases (PTP-1B) belong to the family of Protein Tyrosine phosphatases, maintain the Tyrosine Protein phosphorylation balance together with Protein Tyrosine Kinases (PTK), participate in signal transduction of cells, and regulate the growth, differentiation, metabolism, gene transcription, immune response and the like of the cells. PTP-1B negatively regulates insulin signaling through dephosphorylation of phosphorylated tyrosine residues of Insulin Receptor Kinase (IRK) or IRK active fragments, and can effectively treat type 2 diabetes through inhibition of PTP 1B.
Alpha-glucosidase inhibitors are a class of novel antidiabetic drugs with a sugar structure, and currently, acarbose, voglibose and miglitol are clinically applied. Is widely applied to clinic as a first choice medicine for treating type 2 diabetes and an auxiliary medicine for treating type 1 diabetes. Alpha-glucosidase is located in epithelial cells of brush border membrane of small intestine, and it can hydrolyze disaccharide such as sucrose, maltose, etc. into monosaccharide which can be absorbed by small intestine, and is key enzyme for hydrolyzing carbohydrate in food. Therefore, the inhibition of the enzyme can delay the digestion of carbohydrate, reduce the absorption of glucose into blood, further inhibit postprandial hyperglycemia, improve the control of blood sugar, relieve hyperinsulinemia and simultaneously improve the glucose tolerance, thereby achieving the aim of preventing and treating diabetes and complications thereof.
The invention directly screens protein tyrosine phosphatase (PTP-1B) and alpha-glucosidase as targets. To date, there is no literature record or research report on the application of licorice extract containing glabridin as main active component in treating diabetes. The glycyrrhiza glabra has a long history of folk medication and has long-term stable clinical application, so the glycyrrhiza glabra has a wide development and application prospect.
Disclosure of Invention
The invention aims to provide a preparation method and application of a glycyrrhiza glabra extract, the method comprises the steps of carrying out reflux extraction on roots of glycyrrhiza glabra or glycyrrhiza glabra dregs after glycyrrhizic acid extraction by using an organic solvent ethanol, separating and purifying macroporous resin, settling petroleum ether and ethyl acetate to obtain effective components for reducing blood sugar from glycyrrhiza glabra and clarify the medical application of the effective components, and preliminary activity screening tests prove that: the glycyrrhiza glabra extract obtained by the method has the inhibition effect on protein tyrosine phosphatase 1B (PTP-1B) and alpha-glucosidase, and can be used as a raw material or an auxiliary agent for preparing medicines or health care products for preventing and treating diabetes.
The preparation method of the glycyrrhiza glabra extract comprises the following steps:
a. pulverizing root of Glycyrrhiza glabra Linne or Glycyrrhiza glabra Linne residue after extraction of glycyrrhizic acid, extracting with 80-95% ethanol water solution at 50-80 deg.C for 1-3 times (each for 1-3 hr), filtering, mixing filtrates, concentrating under reduced pressure, adjusting alcohol content to 30-50%, and filtering to obtain alcohol extract;
b. b, adding the alcohol extract obtained in the step a into a pre-prepared macroporous resin column with the model of HPD-300, HPD-400, HPD-600, AB-8 or D101, eluting with 30-50% ethanol, then eluting with 70-85% ethanol with the volume of 3-8 times of the column volume, collecting 70-85% ethanol eluate, and concentrating to obtain a resin purified product;
c. and c, extracting the resin purified product obtained in the step b with ethyl acetate for 1-3 times to obtain an ethyl acetate extract, adding petroleum ether with the volume of 3-5 times that of the ethyl acetate for settling treatment for 1-3 times, collecting supernatant, concentrating and drying to obtain the glycyrrhiza glabra extract with the glabridin content of 40-70%.
The glycyrrhiza glabra extract obtained by the method plays a role in screening a target point protein tyrosine phosphatase 1B of a medicament for inhibiting diabetes.
The glycyrrhiza glabra extract obtained by the method plays a role in screening a target point alpha-glucosidase of a medicament for inhibiting diabetes.
The application of the glycyrrhiza glabra extract obtained by the method in preparing a medicament for reducing blood sugar.
The application of the glycyrrhiza glabra extract obtained by the method in preparing health products or other functional foods.
Compared with the prior art, the preparation method of the glycyrrhiza glabra extract has the following advantages and effects:
(1) the content of glabridin in the glycyrrhiza glabra extract obtained by the method is 40-70%, and the production method is simple and feasible and is suitable for industrial production.
(2) Preliminary activity screening tests prove that the extract has obvious protein tyrosine phosphatase 1B (PTP-1B) and alpha-glucosidase inhibition effects, wherein the alpha-glucosidase inhibition activity is 200 times of that of a positive drug acarbose, and meanwhile, the activity of the extract is superior to that of glabridin with the content of 90%.
Drawings
FIG. 1 is a high performance liquid phase diagram of a glabridin reference substance of the present invention;
FIG. 2 is a high performance liquid phase diagram of Glycyrrhiza glabra extract of the present invention.
Detailed Description
Example 1
a. Crushing 10kg of the root of glycyrrhiza glabra, adding 80% ethanol with 8 times volume concentration, heating and refluxing for 3 times, heating at 50 ℃, for 1 hour, filtering, combining extracting solutions, concentrating under reduced pressure, adding alcohol until the alcohol content is 50%, and filtering to obtain an alcohol extract;
b. b, adding the alcohol extract obtained in the step a into a pre-prepared AB-8 macroporous resin column, eluting with 50% ethanol with the volume concentration 6 times that of the column, eluting with 80% ethanol with the volume concentration 5 times that of the column, collecting ethanol eluate, and concentrating under reduced pressure to obtain a resin purified product;
c. and c, extracting the resin purified product obtained in the step b with 0.3L of ethyl acetate for 2 times to obtain an ethyl acetate extract, adding 1.8L of petroleum ether for settling treatment for 2 times, collecting supernate, concentrating and drying to obtain 30g of the glycyrrhiza glabra extract with the glabridin content of 63%.
Example 2
a. Crushing 10kg of the root of glycyrrhiza glabra, adding ethanol with the volume concentration of 8 times of 85% into the crushed root, heating and refluxing for 3 times, at the temperature of 60 ℃, for 1 hour, filtering, combining extracting solutions, concentrating under reduced pressure, adding alcohol until the alcohol content is 40%, and filtering to obtain an alcohol extract;
b. b, adding the alcohol extract obtained in the step a into a pre-prepared HPD-300 macroporous resin column, eluting with 40% ethanol with 4 times of column volume concentration, eluting with 70% ethanol with 4 times of column volume concentration, collecting ethanol eluate, and concentrating under reduced pressure to obtain a resin purified product;
c. and c, extracting the resin purified product obtained in the step b with 0.4L of ethyl acetate for 3 times to obtain an ethyl acetate extract, adding 3.6L of petroleum ether for settling treatment for 1 time, collecting supernate, concentrating and drying to obtain 25g of the glycyrrhiza glabra extract with the glabridin content of 51%.
Example 3
a. Crushing 10kg of glycyrrhiza glabra dregs after extracting glycyrrhizic acid, adding 6 times volume concentration of 95% ethanol, heating and refluxing for 1 time at 50 ℃ for 3 hours, filtering, concentrating under reduced pressure, adding water until the alcohol content is 30%, and filtering to obtain an alcohol extract;
b. b, adding the alcohol extract obtained in the step a into a pre-prepared HPD-400 macroporous resin column, eluting with 30% ethanol with the volume concentration of 3 times of the column, eluting with 85% ethanol with the volume concentration of 3 times of the column, collecting ethanol eluate, and concentrating under reduced pressure to obtain a resin purified product;
c. and c, extracting the resin purified product obtained in the step b with 0.2L of ethyl acetate for 3 times to obtain an ethyl acetate extract, adding 2.4L of petroleum ether for settling treatment for 1 time, collecting supernate, concentrating and drying to obtain 35g of a glycyrrhiza glabra extract with the glabridin content of 68%.
Example 4
a. Crushing 10kg of the root of glycyrrhiza glabra, adding ethanol with the volume concentration of 8 times of 95 percent, heating, refluxing and extracting for 2 times, at the temperature of 80 ℃ for 2 hours, filtering, concentrating under reduced pressure, adding water until the alcohol content is 40 percent, and filtering to obtain an alcohol extract;
b. b, adding the alcohol extract obtained in the step a into a pre-prepared HPD-600 macroporous resin column, eluting with 50% ethanol with the volume concentration of 5 times of the column, eluting with 85% ethanol with the volume concentration of 3 times of the column, collecting ethanol eluate, and concentrating under reduced pressure to obtain a resin purified product;
c. and c, extracting the resin purified product obtained in the step b with 0.1L of ethyl acetate for 2 times to obtain an ethyl acetate extract, adding 1L of petroleum ether for settling treatment for 3 times, collecting supernate, concentrating and drying to obtain 32g of the glycyrrhiza glabra extract with the glabridin content of 43%.
Example 5
a. Crushing 10kg of glycyrrhiza glabra dregs, adding 10 times of ethanol with the volume concentration of 85% into the crushed glycyrrhiza glabra dregs, heating and refluxing for 2 times, at the temperature of 50 ℃, for 2 hours, filtering, concentrating under reduced pressure, adding water until the alcohol content is 45%, and filtering to obtain an alcohol extract;
b. b, adding the alcohol extract obtained in the step a into a pre-prepared D101 macroporous resin column, eluting with 45% ethanol with 4 times of column volume concentration, eluting with 80% ethanol with 6 times of column volume concentration, collecting ethanol eluate, and concentrating under reduced pressure to obtain a resin purified product;
c. and c, extracting the resin purified product obtained in the step b with 0.2L of ethyl acetate for 2 times to obtain an ethyl acetate extract, adding 1.2L of petroleum ether for settling treatment for 2 times, collecting supernate, concentrating and drying to obtain 28g of the glycyrrhiza glabra extract with the glabridin content of 61%.
EXAMPLE 6
The method for measuring the content of glabridin in the glycyrrhiza glabra extract comprises the following steps:
any one of the glycyrrhiza glabra extracts obtained in examples 1 to 5 was subjected to glycyrrhiza glabra content determination under the following chromatographic conditions: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-water solution (46:54) is used as a mobile phase; the detection wavelength is 282 nm;
preparation of control solutions: precisely weighing glabridin reference, adding anhydrous ethanol to obtain solution containing 40 μ g per 1 ml;
preparation of a test solution: placing a glycyrrhiza glabra extract sample of 30mg in a 25mL volumetric flask, dissolving with absolute ethyl alcohol and diluting to a scale, precisely measuring 2mL of solution, placing in the 25mL volumetric flask, adding absolute ethyl alcohol to dilute to the scale, and shaking up to obtain the glycyrrhiza glabra extract; content determination: precisely absorbing 10 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring, and calculating the content of glabridin in the Glycyrrhiza glabra Linne extract to be 40-70%.
Example 7
Biological activity screening of the use of the glycyrrhiza glabra extract obtained in examples 1 to 5 as a drug or health product of a protein tyrosine phosphatase inhibitor and an α -glucosidase inhibitor:
protein tyrosine phosphatase 1B (PTP-1B) screening method: the activity of protein tyrosine phosphatase 1B is determined by detecting the change of the product on PNPP by using p-nitrophenyl disodium phosphate (pNPP) as a substrate. The total reaction volume was 200. mu.L: 179 mu L of phosphate buffer solution containing PTP1B protein solution, 1 mu L of sample to be tested, 20 mu L of 35mmol/L PNPP, shaking and mixing uniformly, reacting for 30min at room temperature, determining the absorption value at 405nm, using a system without enzyme solution as a blank, and 3- (3,5-Dibromo-4-hydroxy-benz-oyl) -2-ethyl-benzofuran-6-sulfonic acid- (4- (thiazol-2-ylsulfamoyl) -phenyl) -amide as a positive control.
The inhibition rate (I%) [ (active group-active group) ]/[ (active group-active group) ]/(100%), IC50 was calculated with software.
The alpha-glucosidase screening method comprises the following steps: the PNPG is used as a substrate in the experiment, and the activity of the alpha-glucosidase is determined by detecting the change of a product p-nitrophenol (PNPG). The total reaction volume was 100 μ L: 2 mu L of sample to be tested, 71.5 mu L of phosphate buffer solution with pH 6.8 and 1.5 mu L of alpha-glucosidase, incubating for 10min at room temperature, adding 25 mu L of 5mmol/L PNPG, shaking and mixing uniformly, reacting for 30min at 37 ℃, and determining the absorption value at 405nm, wherein a non-enzyme solution system is used as a blank, and acarbose is used as a positive control.
The formula of the inhibition rate (I%) is [ enzyme activity group- (medicine group-medicine control group)/(enzyme activity group-enzyme activity control group) ] × 100%
IC of Glycyrrhiza glabra extract on PTP1B inhibitor and alpha-glucosidase inhibitor50Concentration of
Figure BDA0003333811710000051
And (4) conclusion:
experimental results show that the glycyrrhiza glabra extract obtained by the method has the inhibition effects of a protein tyrosine phosphatase 1B inhibitor and alpha-glucosidase.

Claims (5)

1. A preparation method of a glycyrrhiza glabra extract is characterized by comprising the following steps:
a. pulverizing root of Glycyrrhiza glabra Linne or Glycyrrhiza glabra Linne residue after extraction of glycyrrhizic acid, extracting with 80-95% ethanol water solution at 50-80 deg.C for 1-3 times (each for 1-3 hr), filtering, mixing filtrates, concentrating under reduced pressure, adjusting alcohol content to 30-50%, and filtering to obtain alcohol extract;
b. b, adding the alcohol extract obtained in the step a into a pre-prepared macroporous resin column with the model of HPD-300, HPD-400, HPD-600, AB-8 or D101, eluting with 30-50% ethanol, then eluting with 70-85% ethanol with the volume of 3-8 times of the column volume, collecting 70-85% ethanol eluate, and concentrating to obtain a resin purified product;
c. and c, extracting the resin purified product obtained in the step b with ethyl acetate for 1-3 times to obtain an ethyl acetate extract, adding petroleum ether with the volume of 3-5 times that of the ethyl acetate for settling treatment for 1-3 times, collecting supernatant, concentrating and drying to obtain the glycyrrhiza glabra extract with the glabridin content of 40-70%.
2. The use of the Glycyrrhiza glabra extract obtained by the method according to claim 1 in screening of a drug for inhibiting diabetes mellitus, namely target protein tyrosine phosphatase 1B.
3. The effect of the glycyrrhiza glabra extract obtained by the method according to claim 1 on inhibiting a diabetes drug screening target point alpha-glucosidase.
4. Use of the extract of glycyrrhiza glabra obtained by the method according to claim 1 for the preparation of a medicament for the treatment of hyperglycemia.
5. Use of the glycyrrhiza glabra extract obtained by the method according to claim 1 in the preparation of health products or other functional foods.
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CN114685523A (en) * 2022-04-08 2022-07-01 南京工业大学 Method for extracting glabridin from glycyrrhiza glabra residues and separating and purifying glabridin

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